L £ 3 fi>? ? • / STAPHYLOCCUS EUTEROTOXIU by Agnes E. Mehling A thesis submitted i n p a r t i a l f u l f i l m e n t of the requirements f o r the degree of Master of Arts i n the department of Bacteriology and Preventive Medicine The University of B r i t i s h A p r i l , 1950 Columbia STAPHYLOCOCCUS ENTEROTOXBT The constituents of a synthetic medium and the conditions required "by staphylococcus s t r a i n 12069 alpha for good toxin production have been determined. Because e s s e n t i a l l y a l l the components of the medium are dialyzable, d i a l y s i s f o r 24 hours was found to produce an average reduction of 98.34 per cent of the s o l i d s , but an average reduction of 53 per cent of the potency. Various methods of concentrating the toxin were studied. Vacuum d i s t i l l a t i o n experiments indicated an a l - most complete loss of potency. "Desi-vac" treatment pro- duced a loss of approximately 50 per cent, and a product which varied i n weight content, s t a b i l i t y , t o x i c i t y , and response to p u r i f i c a t i o n . I y o p h i l i z a t i o n produced a loss of approximately 20 per cent, and a product which reacted more consistently to further treatment. The freezing meth- od of concentration was shown to be applicable to the problem. The concentrated toxic f i l t r a t e s were subjected to additional p u r i f i c a t i o n procedures. The toxin was ad- sorbed from solution by "Horit", but i t s elution was not achieved. Sthanol, methanol, and acetone p r e c i p i t a t i o n experiments were performed, but only the methanol treatment showed any evidence that would warrant futther i n v e s t i g a t i o n . Cadmium chloride p r e c i p i t a t i o n removed the active component from f i l t r a t e s , hut residual cadmium proved too toxic in animal tests to permit the degree of separation of enterotoxin to be determined. The probable polysaccharide nature of enterotoxin was demonstrated by enzyme experiments upon crude toxic f i l t r a t e s , but further study was prevented because of the extremely small y i e l d s of r e l a t i v e l y p u r i f i e d material. Enterotoxin p u r i f i e d by acid p r e c i p i t a t i o n at pH'3.5 gave a negative Biuret and a p o s i t i v e Molisch reaction. The low antigenic power demonstrated by the r e l a t i v e l y pure f i l t r a t e s was also i n d i c a t i v e of the non-protein nature of the toxin The acid p r e c i p i t a t i o n at pH 3.5 of dialyzed and concentrated f i l t r a t e s produced a material whose soluble f r a c t i o n was found to have an average weight of 150 gamma per cat test dose, and a nitrogen content of less than 2 per cent. The recovery of enterotoxic potency i n material treated by d i a l y s i s , concentration, and p r e c i p i t a t i o n , averaged 7.33 per cent of the o r i g i n a l . ACKNOWLEDGEMENT I would l i k e to express my appreciation of the encouragement and advice which Dr. Dolman has offerred so generously throughout the course of t h i s research. I would also l i k e to thank the many members of the Department f o r their help and cooperation, without which t h i s work would have been impossible. TABLE OF CONTENTS STAPHYLOCOCCAL ENTEROTOXIN I. Introduction II. Production of Toxin III. IV. A. Preparation of Synthetic Medium B. Preparation of F i l t r a t e s page 1 5 12 P u r i f i c a t i o n of Toxin A. Dialysis 19 B. Concentration Methods 27 C. Precipitation 56 D. Adsorption 59 E. Freezing 59 Nature of Toxin A. Potency 60 B. Non-Protein Properties 61 LIST OF CHARTS Chart I. Chart I I . A summary of the various terms used i n the acid p r e c i p i t a t i o n technique of p u r i f y i n g DDC toxin. 29 The percent of the o r i g i n a l potency of C toxin to he found i n the various f r a c t i o n s involved i n acid precipitations 37 Chart I I I . Summary of experiment 2 : the effect of vacuum d i s t i l l a t i o n on DC toxin 48 LIST OF TABLES TABLE I: The completeness of removal of type C medium hy 42.5 h r s . d i a l y s i s through Visking « No-Jax tubing. , M page 22 T2LBE1 I I : The removal of d i f f u s i b l e materials from type C toxin by d i a l y s i s through Visking "No-Jax" tubing page 22 Ti&BLE I I I : Removal of d i f f u s i b l e materials from type C toxin by d i a l y s i s through Visking »Hb-Jax tubing M TABLE IV: TABLE V: TABLE VI: page 24 Comparison of C and DC toxins with respect to enterotoxic potency. page 25 The r e l a t i o n of the s o l i d content of RS material to cat Test Doses page 31 The r e l a t i o n of the s o l i d content of SP material to cat test doses page 32 TABLE VII; The gradual removal of impurities as revealed by t o t a l solids content per cat test dose. ....page 33 TABLE VIII The per cent of o r i g i n a l DDC s o l i d s found in the RP f r a c t i o n page 33 TABLE IX: TABLE X: TABLE XI: Determination of average percentage of DC cat test doses recovered i n the various f r a c t i o n s page 35 The percentage of the o r i g i n a l C toxin C.T.D.'s detected i n the various fractions page 36 The percent of the o r i g i n a l DC potency recovered i n individual f r a c t i o n s page 38 TABLE XII: A comparison of four bottlesof #26 DDC toxin which had undergone "Desi-vac " treatment. .page 42 TABLE XIII Determination of the average percentage of DC cat test doses i n the various fractions of material treated by l y o p h i l i z a t i o n page 5A TABLE XIV: The percentage of the o r i g i n a l cat test doses i n C toxin detected i n the various f r a c t i o n s of l y o p h i l i z ed material page 52 The percentage of the o r i g i n a l cat test doses i n the various f r a c t i o n s of material concentrated by lyophi l i z a t i o n at the C stage page 53 A comparison of the r e s u l t s obtained from 3 methods of concentrating DC toxin page 55 The recovery and degree of purity of ethanol precipitated toxin compared to SP. • page 57 TABLE XVIII. Milligrams of nitrogen i n the various f i l t r a t e s as revealed by microKjeldahls page 62 TABLE XV: TABLE XVI: TABLE XVII. 1 STAPHYLOCOCCUS ENTEROTOXIN Introduction Staphylococcus enterotoxin i s undoubtedly res- ponsible f o r a greater number of food poisoning outbreaks than any other agent. But, because of the transient nature of the attack, the r a p i d i t y of complete recovery, and the frequency with which such attacks are experienced - t h i s source of great discomfort and economic loss i s often d i s regarded, and only the more dramatic ing outbreaks are noted. types of food poison- Fortunately, i n recent years, there has been a growing awareness of the f a l l a c y of t h i s attitude, and a number of workers have turned their attent i o n to t h i s product of the ubiquitous staphylococci. The study of t h i s toxin was retarded f o r years by the belief that the presence of contaminating l o c o c c i i n foodstuffs was usually i r r e l e v a n t . staphy- The prevalence of these organisms* and the fact that only a few of the strains are capable of producing enterotoxin, may have done much to substantiate t h i s b e l i e f i n the minds of early investigators. Indeed, i t was not u n t i l 1930, when Dack et a l (1) f i r s t reported the administration of f i l t r a t e s to human volunteers* that the p o t e n t i a l importance of these organisms was r e a l i z e d . Moreover, the contentious reports concerning the toxigenic spectrum of staphylococci added greatly to the confusion. The fact that both alpha and beta toxins apparently evoke some gcuteoga-stro.iintest- - 2 - i n a l symptoms found i n enterotoxin poisoning l e d many workers to believe that they were dealing with onejsubstance. As recently as 1943, Pulton (2) reported the inseparabili t y of enterotoxin and alpha-toxin, and i n 1939, Kojima and Kodama (3)^and i n 1941, Woodward and Slanetz (4) i d e n t i f i e d enterotoxin with beta-toxin. However, c e r t a i n physical and b i o l o g i c a l propert i e s have now established the f a c t that enterotoxin i s a d i s t i n c t entity, and that i t i s only c o i n c i d e n t a l that many food poisoning strains produce alpha-orbetaas w e l l . The comparative thermostability and low antigen- i c properties are c h a r a c t e r i s t i c s of enterotoxin. thst toxins The f a c t i t s e f f e c t s are not n u l l i f i e d by antisera which neut- r a l i z e alpha-and beta-toxins seems conclusive. a staphylococcus Furthermore, s t r a i n has been found which produces alpha- and enterotoxin to a high t i t r e under onejset of conditioss, but only n e g l i g i b l e traces of alpha-toxin, and enterotoxin to i t s former l e v e l , under altered conditions. These pec- u l i a r properties, and others which w i l l be revealed i n the course of t h i s report, are indicative of the unique posi t i o n that staphylococcus enterotoxin should be assigned. In addition to the contentious nature of early findings i n t h i s f i e l d , the problem of testing f o r enterotoxin has always presented a handicap to the i n v e s t i g a t i o n of t h i s subject. In spite of numerous attempts, no s a t i s - factory method f o r determining t o n i c i t y has been found 3 which does not involve the pse of some laboratory animal usually the cat, although i n some cases, resort has been made to monkeys, or even humans* Thus, because of the p a r t i c u l a r l y unpleasant effects of enterotoxin, the indefiniteness of the reaction which involves long hours of caref u l observation, the aesthetic barriers presented by the usual cat t e s t , and the d i f f i c u l t i e s experienced i n the maintenance of a healthy supply of these animals - i t i s not surprising that investigations of t h i s subject have been r e l a t i v e l y rare. It i s hoped that with continued study of the problem, s i m p l i f i e d tests w i l l be evolved. Once i t i s r e a l i z e d that enterotoxin exerts such an important and unavoidable influence on our d a i l y l i v e s , the necessity becomes apparent f o r data concerning the requirements of toxin production. If these be known, improved handling of food, with the elimination of a l l possible opportunities f o r enterotoxin formation, may result i n a happier enjoyment of a greater variety of foodstuffs. Obviously, i n an investigation of t h i s problem, one of the requirements f o r further study i s the development of a large-scale method the t o x i n . of producing and p u r i f y i n g As w i l l be apparent from t h i s report, the extreme d i l u t i o n of the potent factor as compared with oth- er toxins which have been p u r i f i e d , poses one of the most d i f f i c u l t problems of p u r i f i c a t i o n ; and u n t i l a dependable method, adaptable to large-scale production has been dev- 4 i s e d , continued 1 r e s e a r c h w i l l be d i f f i c u l t . In summary, the purpose of t h i s i n v e s t i g a t i o n has been: 1) to d i s c o v e r a d d i t i o n a l f a c t s concerning the nature of e n t e r o t o x i n , so t h a t f u r t h e r proof as to i t s p o s i t i o n as a distinct entity w i l l e x p l a n a t i o n may be a v a i l a b l e , and so t h a t a p o s s i b l e be found f o r i t s low a n t i g e n i c powers and i t s s p e c i f i c a t t a c k on one c e n t r e of the c e n t r a l nervous system. 2) to study the c o n d i t i o n s of t o x i n p r o d u c t i o n a b e t t e r understanding may be so t h a t of e s s e n t i a l food c o n t r o l problems obtained.. 3) to d e v i s e a method by which e n t e r o t o x i n can be pro- duced and p u r i f i e d oh a l a r g e s c a l e so t h a t i t s f u r t h e r study w i l l be enhanced. I I . PRODUCTION Off TOXIN Throughout t h i s i n v e s t i g a t i o n staphylococcus s t r a i n 12069 alpha was to be c o n s i s t e n t l y alpha- and on proteose at employed. T h i s organism has enterotoxigenic. proven When grown peptone, s e m i s o l i d agar medium f o r 40 hours 37°C. i n an atmosphere c o n t a i n i n g 30 per cent carbon d i o x i d e , t h i s s t r a i n produces both t o x i n s ; however, when grown on the same medium at room temperature, only neg- l i g i b l e amounts of a l p h a t o x i n , but equal t i t r e s of o t o x i n , are produced. (5) Thus, t h i s p a r t i c u l a r enter- staphylo- 5 coccus s t r a i n affords a most suitable source of enterotoxin. By t h i s simple switch of incubation temperatures, the f i r s t step of p u r i f i c a t i o n can be achieved; namely, the reportedly impossible separation of enterotoxin from alpha t o x i n . Whenever, during the course of experimental work, nontoxic f i l t r a t e s were obtained - the organism was always rechecked for possible v a r i a t i o n by growing i t again on t h i s soft agar medium, and at no time did negative filtrates r e s u l t under these conditions. A. Preparation of Synthetic Medium As soon as the study of enterotoxin was undertaken, the necessity for a synthetic medium became apparent. chemically reproduceable medium was essential i n order that consistent toxin production could be assur/ed. of greater importance was A But the fact that a less comples med- ium would simplify greatly the p u r i f i c a t i o n procedure. The advantage of having the constituents of the medium as unrelated as possible i n molecular size and behaviour to the material to be p u r i f i e d was recognized. ly work concerning was From ear- the possible nature of enterotoxin, i t evident that i f t h i s substance were a protein, i t was probably of the molecular size of the proteoses (6); that i t might even be a complex carbohydrate (7). the i n a d v i s a b i l i t y of the continued use of peptone semi-solid agar was but Thus proteose obvious. The n u t r i t i o n of staphylococci i n general, has 6 been studied i n considerable d e t a i l , and a perusal of the l i t e r a t u r e produced an excellent background of information. The work of F i l d e s , Richardson, Knight, and Gladstone ( 8 ) revealed the e s s e n t i a l i t y of the following amino acids for the rapid growth of staphylococci: arginine, aspartic a c i d , cystine, glycine, h i s t i d i n e , leucine, proline, and v a l i n e . phenylalanine, Lysine, methionine, and tyrosine were of variable importance, and tryptophane was required by some s t r a i n s . The only essential vitamins according to Knight (9) were thiamin and n i c o t i n i c a c i d , but Porter and Pelczar (10) reported b i o t i n to be required by c e r t a i n strains. The necessity of u r a c i l f o r anaerobic growth was noted (11). F i l d e s , et a l (8) found that ferrous i r o n and magnesium s a t i s f i e d the mineral requirements, that d-glucose furnished a convenient carbohydrate source, and that phosphate served as an excellent buffering agent. After preliminary work u t i l i z i n g the s i m p l i f i e d medium of Favorite and Hammon (12) and a medium designated "type M", Casselman (13) devised a synthetic medium (type C) f o r use i n the study of enterotoxin. A l l the prelimin- ary experiments involved i n the evolution of this medium concerned themselves only with maximum growth, and d i s r e garded the subject of maximum toxin production. Because l a t e r work has shown that t o x i c i t y does not necessarily p a r a l l e l growth, i t i s quite possible that by attention to t h i s l a t t e r f a c t , a medium could be devised which would r e s u l t i n greater toxin production. However, d i f f i c u l t i e s involved i n the testing f o r toxin injthe innumerable possi b i l i t i e s * and the fact that the toxin l e v e l on the "C" medium was o r i g i n a l l y found to be almost as high as on the soft agar - l e d to the acceptance of type "C" medium as the basis f o r enterotoxin production by s t r a i n 12069 alpha. But**, the frequent appearance of slimy or "rough « growth, the lowering of potency l e v e l s to l e s s than h a l f of e a r l i e r ones from time to time, and the occasional period when f i l t r a t e s revealed no t o x i c i t y at a l l , suggested that type "C" medium does not enhance the s t a b i l i t y of enterotoxigenic properties of t h i s organism. At the beginning of t h i s research, t h i s type C medium was prepared exactly as outlined by Casselman, but l a t e r , because of reasons to be detailed shortly, minor modifications were made. A summary of the preparation of C medium follows. A. Chemicals and Solutions Required. 1. Solution of Bacto-Casamino Acids prepared accord- ing to procedure on next page. * - 2 . Bacto-dextrose i n d i s t i l l e d water, 50$ 3 . Potassium dihydrogen phosphate ( C P . ) i n d i s t i l l e d water, 10$ 4. Sodium hydroxide (C.P.) i n d i s t i l l e d water, 10$ * 5. Nicotinic acid i n d i s t i l l e d water, 0.5% *" 6 . Thiamin hydrochloride i n d i s t i l l e d water, 3 . 3 $ * 7. U r a c i l i n d i s t i l l e d water, 1.0$ ** 8. Mineral mixture prepared, "by d i s s o l v i n g the following C.P. s a l t s i n 1 l i t r e of d i s t i l l e d water and adjusting the mixture to pH 3.0 or lower with concentrated HCl MgSOy.. '7E Q 20 gms PeSO . 711^0 6.0 gms MnSO^.^H^O 2.0 gms K f 9. Disodium phosphate (C.P.), s o l i d * These solutions were s t e r i l i z e d hy autoclaving i n large f l a s k s at 240 P. f o r 10 mins. B. Preparationbf Solution of Casamino Acids. A solution of 500 gms of Bacto-Casamino Acids i n 5 l i t r e s of distilledjwater was prepared and adjusted to pH 3.5 with concentrated hydrochloric a c i d . Approximately 100 gms. of E o r i t - A was added to the a c i d i f i e d solution, and the mixture was s t i r r e d w e l l to disperse the decolorizing charcoal. After being allowed to stand f o r 20 minutes i t was f i l t e r e d through Whatman's No. 1 f i l t e r paper on a Buchner funnel. I f the filtrate;, was at a l l coloured, the treatment with E o r i t - A was repeated after rechecking the pH. To the colourless f i l t r a t e were added: arginine hydrochloride 15.0 gms. cystine 3.5 gms. glycine 20.0 gms. tryptophane 2.5 gms. The mixture was stored i n suitable f l a s k s i n the r e f r i g e r - ator. C. Preparationjof Medium, part (1) solution of Bacto-Casamino Acids 1000 ml. d i s t i l l e d water 3750 ml. n i c o t i n i c acid uracil (0.5%) (1.0%) NaOH (10%) 2.5 ml. 2.5 ml. - to pH 7.4 part (2) d i s t i l l e d water Na^HPO^ .12HjO 3750 ml. 200 gms. warmed to dissolve . part (3) mineral mixture thiamin HCl (3.3$) 34 ml. 2 ml. prepared with aseptic precautions 1) Parts (1) and (3) were mixed and adjusted to pH 7.4 with 10$ KH^PO^. 2) S u f f i c i e n t water was added to make the volume up to 9950 ml. 3) I f necessary, the mixture was readjusted to pH 7.4 with 10$ KH^PO^. or 10$ NaOH and then the volume was made up to 10.0 l i t r e s . 4) This mixture was dispensed o r i g i n a l l y 1 l i t r e per gallon b o t t l e and autoclaved at 252 P. f o r 20 mins. 5) The following were added to each bottle when cool a i d 10 before inoculation: part (3) 50$ d-glucose 3 ml. 20 ml. It i s i n t e r e s t i n g to note the s i m i l a r i t y between t h i s type C medium and that devised by Surgalla (14 & 15) for enterotoxin production. The ingredients are much the same, the major difference being h i s use of i n d i v i d u a l amino acids throughout; whereas, type C medium u t i l i z e s Bacto-Casamino acids as i t s base. Moreover, i n general, the concentrations of the substanc es are higher i n C medium, and a few notable examples follow: thiamin i s added i n approximately 200 times the quantity Surgalla uses; glucose, 5 times; cystine, 5 t i m e s ; and so on. The inclusion of manganese i n type C medium i s another d i f f e r e n t i a t i n g point. Unfortunate^ , consistently potent batches of enterotoxin were not obtained, and Wood (16) reported negative cat tests i n quantities up to 2.5 c c . f o r some 6 weeks before the problem was taken over by t h i s worker. When 2 additional batches o f . f i l t r a t e produced negative cat reactions i n quantities up to 3.0 c c , attention was turned again to the constituents of the medium and the conditions of growth. It was r e a l i z e d that v a r i a t i o n of the organism i t s e l f might be involved, but when f i l t r a t e s prepared on proteose-peptone, semi-solid agar yielded the usual potenc- ies of enterotoxin - t h i s p o s s i b i l i t y was d i s c r e d i t e d . 11 The v a r i a t i o n was one which developed only when the organism was grown on C medium. At a l l times* growth i n the non-toxic cultures was just as abundant as i t had been i n toxic ones. Because the switch" from 4 per cent to 2 per cent disodium phosphate had been made by Casselman (13) just p r i o r to the time when the non-toxic f i l t r a t e s were obtained, t h i s factor was investigated f i r s t . But the increase of the phosphate content to the former 4 per cent value produced no detectable difference i n the f i l t r a t e s obtain- ed. Next followed an i n v e s t i g a t i o n of the minerals used. Was the i n c l u s i o n of MnS0 i n the medium j u s t i f i e d when 4 one considered the former acceptance of the report of F i l d e s , Richardson, Knight, and Gladstone (8) that "the mineral requirements of the staphylococci are s a t i s f i e d by ferrous i r o n and magnesium," and the subsequent f i n d i n g of Surgalla (15) that enterotoxin i s produced on a synthetic medium containing only these 2 minerals? Could the manganese be exerting an inhibitory effect on enterotoxin production by 12G69 alpha under c e r t a i n conditions? In h i s report (13), Casselman gave no explanation f o r i t s i n c l u s i o n . However, when 12G69 alpha was grown on C medium, complete i n every way except f o r the omission of manganese sulfate, f i l t r a t e s were s t i l l produced. sonal communications, non-toxic Later, Casselman, i n per- substantiated these findings by 12 stating that, a f t e r the appearance of Wolf's (17) paper, he employed manganese sulfate experimentally times and found that concentrations at various : as high as 0 .G004M (20 times the concentration i n type C medium) had no i n h i b i t o r y effects. The possible importance of b i o t i n was also i n - vestigated, because of the findings of Porter and Pelczar (10) that i t was e s s e n t i a l for the growth of some s t r a i n s of staphylococci, and because of i t s omission from G medium on the basis of growth, not toxin t e s t s . However, the i n - clusion of b i o t i n produced no improvement i n the toxin production. During t h i s study of the possible e f f e c t s of phosphate concentration, the omission of manganese, and the i n c l u s i o n of b i o t i n - not only the potency of the f i l t r a t e s , but also the degree of growth, c o l o n i a l c h a r a c t e r i s t i c s of the organisms when plated out, and the pH of the f i l t r a t e s , were investigated. However, at no time, was v a r i a t i o n evident. At t h i s point, attention to methods of production any detectable (which w i l l be summarized i n the following sect- ion) resulted i n the production of potent f i l t r a t e s , and the investigation of the effects of medium eonstituents on toxin production was 33. discontinued. Preparation of F i l t r a t e s The preparation of enterotoxic f i l t r a t e s always 13 commenced w i t h the i n o c u l a t i o n of a t y p i c a l c o l o n y of 12069 a l p h a from a 24-hour blood p l a t e i n t o 15 C medium. T h i s was incubated at room temperature growth became evident e n t i r e c u l t u r e was containing ( u s u a l l y 6 h o u r s ) , and inoculated sufficient a g i t a t e d a t the time of i n o c u l a t i o n , and fairly t u r b i d c u l t u r e was that growth was i o n a l l y , and then t h i s cent T h i s f l a s k was well l e f t at room temp- hours - or a t l e a s t u n t i l obtained. Because of the enhanced i f the c u l t u r e was because of the l a t e r d i s c o v e r y ce of a e r a t i o n on e n t e r o t o x i n was of until C medium to p r o v i d e a 2 per u s u a l l y f o r 18-i&o24 cc. i n t o an erlemneyer f l a s k inoculum f o r the batch to be p r e p a r e d . erature or 20 fact shaken occasof the p r o d u c t i o n - the influen- culture c a r e f u l l y swished around from time to time. same reason, an i n o c u l a t i n g c u l t u r e was a Por the never prepared i n a f l a s k which c o n t a i n e d G medium to a depth of more than l-§- inches. At the b e g i n n i n g of t h i s p r o j e c t , the batches of t o x i n were prepared i n 9 l i t r e Pyrex s o l u t i o n b o t t l e s , each c o n t a i n i n g 2 litres of medium. These b o t t l e s , i n a h o r i z o n t a l p o s i t i o n which ensured a l a r g e placed surface a r e a f o r the c u l t u r e , were incubated on the bench top 3-g- days. At the .end of t h i s , time, the c u l t u r e s were ered through # 1 Whatman f i l t e r paper, pooled, and ed by S e i t z f i l t r a t i o n . t a i n e d was The pale yellow f i l t r a t e designated type "C" toxin. for filt- sterilizthus ob- 14 - However, as mentioned e a r l i e r , the f i l t r a t e s prepared according to this o r i g i n a l protocol lacked potency, and so some of the possible reasons were investigated. It was thought that perhaps the organism required a training period i n which to adapt i t s e l f to the new medium, and so i t was passed through a series of 7 subcultures, with a 24 hour incubation period f o r each. From . the blood plate, i t was inoculated into a small f l a s k containing 60 per cent proteose-peptone broth and 20 per cent C medium, then into one containing 60 per cent proteosepeptone broth and 40 per cent C medium,and so on, with 20 per cent increments, u n t i l f i n a l l y i t was growing on 100 per cent C medium. Two additional subcultures were made, and the f i n a l culture was employed f o r the inoculation of one of the large bottles* The r e s u l t i n g f i l t r a t e gave a negative cat reactionjin doses up to 3.5 ml. In order to ensure a young viable culture of 12069 alpha whichcontaire d only organisms that had revealed themselves capable of speedy adaptation to growth i n C medium^ and which might have retained t h e i r toxin-producing a b i l i t y , numerous, rapid subcultures were made. From a 24 hour culture on a blood agar plate, 12069 alpha was inoculated into ^ 20 ml. of C medium. 3?our subcultures were made each day f o r 3 days (that i s , approximately hours during the day) every 3 At the end of t h i s time, a larger glask was inoculated and the regular procedure carried out, but again, no enterotoxin was detected by cat t e s t s . Because other workers (13) had noted the e f f e c t of the volume of medium i n a container upon the potency of toxin, the next possible f a c t o r considered was flask size. that of The use of 1 l i t r e of medium i n a g a l l o n bot- t l e and 800 ml. i n a 2 - l i t r e f l a s k made no detectable dif- ference, although these r a t i o s had previously been found favourable f o r toxin production ML. (16). However, when 300 of C medium i n a 2 - l i t r e erlenmeyer f l a s k was a p o s i t i v e cat reaction was employed, obtained from 2.5 ml. of the resulting f i l t r a t e . Thus, i t became evident that the r a t i o between volume of medium and the surface exposed to the a i r , or i n short, the degree of aeration, was a c r i t i c a l point. Therefore, the former method of the aeration of cultures by r o t a t i o n was resumed. V a c o l i t e r j a r s of the \ l i t r e and l i t r e size were employed with 100 and 200 of' C medium r e s p e c t i v e l y . ml. They were t i e d to^the frame of a b a l l m i l l , and rotated on their long axes. The very f i r s t batch prepared i n t h i s fashion produced a C toxin with a cat M.R.D. of 1.5 ml., and so the necessity f o r aeration was established. Further experiments revealed that the pre- sence of u r a c i l exerted no detectable influence on f i l t r a t e s , and so i t was dropped from the formula f o r C medium. The capacity of the frame, the small amounts of medium which the V a c o l i t e r j a r s would hold i n the horizontal p o s i t i o n ? 16 - and the f a i l u r e of these jars to withstand autoclaving more than 6 or 8 times - necessitated the a l t e r a t i o n of the frame and the procurement of large Pyrex f l a s k s . the present time, the rotating apparatus handles At four 9 - l i t r e f l a s k s containing 2 l i t r e s each; that i s , each batch consists o r i g i n a l l y of 8 l i t r e s of medium - a quantity which seems to u t i l i z e the available f a c i l i t i e s to t h e i r capacity. If future expansion of equipment should enable the handling of larger batches of toxin at one time, the problem of aeration might be handled with greater; f a c i l i t y by bubbling a i r through the cultures. With the adoption of t h i s method, much larger volumes of medium could be dispensed into each f l a s k , and the irksome task of tying the f l a s k s onto a frame would be eliminated. However, unless the available a i r pressure was controlled with greater r e g u l a r i t y than during the past year, i t s u t i l i z a t i o n would lead to complications. The use of tanks of compress- ed a i r would provide a more r e l i a b l e flow, but the expense involved might present a serious handicap. To summarize t h i s aeration problem, one may say that, from time to time, the staphylococcus s t r a i n 12069 alpha seemed to undergo some inherent change which rendered i t incapable of producing enterotoxin under previously favourable conditions. The r e s u l t s obtained suggested that the need f o r aeration was not completely eliminated by the 17 - use of u r a c i l , and that t h i s s i m p l i f i c a t i o n i n enterotoxin production was inadvisable. Shortly a f t e r the resumption of the r o t a t i o n method of aeration, an apparent change was noted i n the growth c h a r a c t e r i s t i c s of 12069 alpha. At the end of 3^- days, the culture f l u i d was a d i s t i n c t yellow; whereas, when grown by the bench top method (with u r a c i l ) , the cultures became, at most, a pale cream. Moreover, the v i s c o s i t y of the cul- tures seemed to increase, growth was very heavy, and the resultant f i l t r a t e was of a syrupy consistency. This l a t - ter change did not occur immediately, but seemed to develop gradually over a period of time with subsequent batches. , It could be correlated neither with any changes i n the comp o s i t i o n of the medium, nor with any a l t e r a t i o n s i n preparation. At one time, i t was thought that there might be some r e l a t i o n between the increasing amounts of sunlight and the "sliminess" of the growth; however, subsequent, batches grown during 3^- days of d u l l l i g h t , when bright sunshine was n o t evident, were just as turbid as those grown during days which included bright sunshine. About a year ago, the organism began t o display another growth v a r i a t i o n on G medium. During the f i r s t one, o r even two, days o f incubation on the rotator - i t assumed a flakey growth which gradually resolved itself into a heavy, granular, and f i n a l l y a thick rope stage. large clumps of, growth could be dispersed with vigorous The 18 - shaking, hut after a few.^ minutes of continued rotation, they would again "begin to aggregate u n t i l , within an hour or so, the s i t u a t i o n would he much as before. plausible explanation could he o f f e r r e d . Again, no Of course from time t o time, new bottles of Bacto-Casamino acids, glucose, etc., had to be opened, hut never did any recorded change in growth seem to coincide with the u t i l i z a t i o n of these "new ingredients. 11 During a l l these degrees ofjdhange, there were no c o l o n i a l variations of 12069 alpha as revealed by growth on blood plates, there was no change i n the production of alpha-and enterotoxin on proteose-peptone semi-solid agar, there was no evidence of bacteriophage a c t i v i t y on plates, and there was no marked reduction i n the enterotoxin potency of the C f i l t r a t e s , but there did sesm to be a growing tendency for 2.0cc. rather than 1.5 cc. to constitute the M. R. D. dose. Unfortunately, the increased v i s c o s i t y of the c u l tures did much to hamper the ease with which C f i l t r a t e s could be obtained. The preliminary f i l t r a t i o n of the c u l - ture f l u i d through # 1 Whatman f i l t e r paper no longer proved s u f f i c i e n t treatment p r i o r to the Seitz f i l t r a t i o n . This procedure was, at best, never a rapid one, but with the increasing v i s c o s i t y , i t became v i r t u a l l y impossible. The pad on a 2 - l i t r e Seitz would become completely blocked after less than 1 l i t r e of culture had been s t e r i l i z e d . The 19 use of c l a r i f y i n g pads p r i o r to s t e r i l i z a t i o n , the c e n t r i fugation of the culture f l u i d , the a d d i t i o n a l use of f i l t e r paper, cheese c l o t h , etc., and the use of increased pres- sure (15 pounds) which necessitated the i n c l u s i o n of a second EK pad, were a l l investigated with varying degrees of success. The c l a r i f y i n g pad treatment, contrary to the early indications, now seems to he of l i t t l e value. At present, the most successful method seems to consist of the following: f i l t r a t i o n of culture through No. 1 Whatman f i l t e r paper, centrifugation of turbid suspension i n 250 ml. cent- rifuge cups f o r -g- hour at 200 r.p.m., and Seitz f i l t r a t i o n . Even with this treatment, the frequently pads have ing the operation. process to be changed i s a slow one, and a number of times dur- It i s not surprising that t h i s stage of the preparation of enterotoxin constitutes a r e a l bottle neck; It i s suggested that possible c l a r i f i c a t i o n of the suspension by some adsorbing agent such as charcoal be investigated. should The readiness with which enterotoxin i s adsorbed by such treatment at l a t e r stages of p u r i f i c a t i o n does not augur well for t h i s method, but the p o s s i b i l i t y that some adsorbing agent might be found with a preferent- i a l action on unwanted impurities should not be d i s c r e d i t - ed. III. PURIFICATION Off TOXIN A. D i a l y s i s One of the p r i n c i p a l considerations behind the development of C medium was that i t s constituents be d i a l y z - 20 - able, and because t h i s s i t u a t i o n was fundamentally achieved, i t i s not surprizing that the f i r s t p u r i f i c a t i o n of G toxin should be d i a l y s i s . step i n the In t h i s way not only the constituents of the medium, but also c e r t a i n dialyzable products of b a c t e r i a l growth can be removed. Cold running tap water was found to be a suitable d i a l y z i n g agent (12) A 10 gallon granite-ware p a i l was used, the l e v e l and inflow of water were controlled by a f l o a t value, and the outflow was achieved by 2 suction tubes. By means of t h i s apparatus, approximately 6 l i t r e s of water were allowed to flow i n and out of the tank each minute. The C toxin was dispensed i n approximately 500 ml. quantities into the d i a l y z i n g tubing (Visking c e l l u l o s e sausage tubing, "No-Jax", 36/32). This tubing, cut i n 3 metre lengths, was knotted at one end, and attached at the other to a rubber stopper carrying a vent tube and a small funnel to f a c i l i t a t e f i l l i n g . This tubing was then clamped by both ends to a rod fixed above the tank - so that the f i l t r a t e was suspended i n the form of a "U". In t h i s way, up to 8 l i t r e s of C toxin could be dialyzed at one time. At the end of 24 hours ( the customary dialyz- ing time), the material was removed from the tank, pooled, and s t e r i l i z e d by Seitz f i l t r a t i o n . of dialyzed C toxin was designated DC M The s t e r i l e filtrate toxin". Casselman (12) i n a study of the e f f i c a c y of d i a l y s i s i n the removal of medium constituents obtained r e s u l t s - 21 - that indicated the " e s s e n t i a l l y complete" discharge of C medium. (Table # 1 constitutes a t y p i c a l i l l u s t r a t i o n of this fact.) O r i g i n a l l y , Casselman (12) concluded, from a study of the effect of time of d i a l y s i s upon the removal of t o t a l solids ( Table # 2), that 24 hours would be d i a l y z i n g time. It was the optimum f e l t that the r e l a t i v e l y small re- duction i n t o t a l s o l i d s obtained by an a d d i t i o n a l 24 hours of treatment was not warranted, because of the danger of contamination and the possible destruction of toxin under such conditions. However, Wood (15) found, i n subsequent studies, that neither of these p o s s i b i l i t i e s was r e a l i z e d by the increased d i a l y s i s period, a.nd that the further removal of impurities, as indicated by t o t a l S3 l i d s , larger than o r i g i n a l experiments indicated. was Thus, at the beginning of this investigation, the 48 hour period employed; but when growth was observed on one or two ions i n the casing, the 24 hour treatment was ed. It was considered was occas- again adopt- that by-products of the growth of contaminants would pose a more serious problem than the minute amounts of d i f f u s i b l e medium constituents that would be removed by prolonged d i a l y s i s . Because complete data regarding the degree of p u r i f i c a t i o n achieved by each processing step was i a l , information concerning was essent- the e f f i c i e n c y of d i a l y s i s c o l l e c t e d for each batch of toxin prepared, and a few 22 TABLE It The completeness of removal of type C medium by 42*5 h r s . d i a l y s i s through Visiting "No-Jax" tubing Before Dialysis Total solids 6.205$ Amino acids ( a ) Reducing sugars (b) Phosphate (c) After Dialysis ' " . Expt. 1 Expt. 2 o .oo4$ 0.002$ v. f t . t r . 0 0 0 tr. Ferrous iron (d) ft. tr. 0 0 a) by triketohydrindene hydrate and by p-dime thy laminobenzaldehyde b) by Benedict's reagent c) by ammonium mo.lybdate and benzidine d) by d i p y r i d y l TABLE l i t The removal of d i f f u s i b l e materials from type C toxin by d i a l y s i s through Visking "No-Jax" tubing. Time of d i a l y s i s 0 hrs. Total solids 1.310 gms. 17 0.028 24 0.011 41 0.008 48 0.008 64 0.010 88 0.009 per 25 ml. sample 23 - of the t y p i c a l r e s u l t s are summarized i n Table 3. t o t a l s o l i d s content was The calculated from r e s u l t s obtained by drying 25 ml. samples of toxin at 37°C. or (after the a r r i v a l of the vacuum oven) at 37"c.in vacuo. The first 4 sets of figures ( l o t s 58-61) represent r e s u l t s obtained by Wood, and i l l u s t r a t e the difference she observed i n the effect of 1 or 2 days d i a l y s i s . The differences recorded by t h i s worker were not so great; i n f a c t , the average percentage removal of t o t a l s o l i d s by 48 hours d i a l y s i s was 99.21$, and by 24 hours - 98.34$ But, i n addition to the degree of p u r i f i c a t i o n accomplished by d i a l y s i s * the percentage recovery of enter- otoxin i s also of prime importance. For the removal of t o t a l s o l i d s by d i a l y s i s would be useless, i f i t were found that the enterotoxin was completely l o s t i n the process. Unfortunately the loss of enterotoxin at t h i s stage of th e p u r i f i c a t i o n i s considerable (Table 4), the average recovery being only 47$. There are a number of possible ex- planations f o r this marked reduction: part of the enterotoxin may be adsorbed onto the c e l l u l o s e casing, part of i t may undergo denaturation during the procedure (either at the time of the actual water treatment, or when the material i s exposed to aeration during f i l l i n g and emptying of the tubing, and during Seitz f i l t r a t i o n ) , part of i t may out through the d i a l y z i n g casing, or part of i t may adsorbed onto the Seitz pad. diffuse be Variations i n the r e s u l t s of - 24 TABLE I I I ; Removal of d i f f u s i b l e materials from type C toxin by d i a l y s i s through Visking "No-Jax" tubing. Lot # Total isolids * DC toxin Time of dialysis C toxin 58 20 hrs. 5 . 3 4 5 gms. 59 221 II 5.265 60 44 II 61 48 1 0.194 gms- reduction 96.37$ n 0.160 II 96.96$ 5.270 II 0.045 II 99.15$ it 5.195 •i 0.029 II 99.14$ 48 n 3.324 II "0.026 •t 99.22$ 2 48 n 3.140 a 0.020 II 99.36$ 3 48 II 2.664 it 0.021 II 99.21$ 4 48 II 2.440 II 0.021 II 99.14$ 5 48 n 2.581 ti 0 . 0 2 2 t; II 99.15$ 15 24 II 3.033 it 0.040 H 98.68$ 16 24 II 3.247 it 0.039 II 98.80$ 17 24 ti 2.802 II 0.036 II 98.72$ 18 24 II 2.870 it 0.034 It 98.82$ 19 24 II 2.886 ii 0.038 H 98168$ 20 24 II 2.565 II 0.050 II 98.05$ 30 24 II 2.773 ii 0.056 II 97.98$ 31 24 II 2.950 n 0.038 II 98.71$ 32 24 II 3.044 ii 0.058 II 98.10$ 33 24 II 2.320 n 0.046 II 98.24$ 34 24 II 2.647 II 0.045 It 98.30$ 35 24 II 2.565 II 0.062 II 97.68$ pe»- IOO m l . 25 TABUS IV; Comparison, of C and DC toxins with ^respect to enterotoxic potency* Lot No Time of Total Volume Dialysis DC Cat Test Doses C Toxin DC Toxin x V o l . Tot. ml. 60 44 hrs. 61 48 62 5.0 L. 7.6 L. 1.5 3330 ti 4.9 L. 6.4 II 1.5 44 ti 4.5 ti 5.2 II 63 46 II 5.3 II 8.5 64 46 it 4.3 N 1 48" 1.9 2 48 II 3 48 4 V o l . Tot. ml. 2170 65$ 3280 3.5 1860 56$ 1.5 3000 3.0 1730 58$ II 1.5 3530 3.5 2430 69$ 6.8 II 2.0 2150 5.0 1360 63$ II 2.3 II 1.0 1875 2.5 925 49.3 % 1.9 II 2.4 it 1.5 1280 3.0 800 62.5 $ II 1.7 II 2.4 it 1.5 1148 4.0 600 52.3 $ 48 •i 1.9 II 2.3 II 2.0 840 4.5 512 62.0 $ 5 48 II 1.0 II 1.4 II 1.2 852 4.0 350 41.1 $ 6 48 it 2.22 II 2.8 II 1.5 1470 4.0 700 47 .6$ 7 72 II 1.7 II 2.3 it 1.3 1340 5.0 465 34.6 $ 8 72 II 1.7 II 2.2 II 1.5 1150 5.0 440 38.2 $ 9 72 II 1.9 II 2.4 it 1.5 1255 5.0 486 38.7$ 10 24 II 2.6 II 3.3 II 1.5 1735 3.5 942 54.3 $ 11 24 II 2.9 It 3.6 it 1.5 1960 4.0 905 46.1 $ 12 24 II 2.8 II 3.1 II 2.0 1400 5.0 629 44.8 $ 13 24 II 2.9 II 3.5 II 2.0 1470 5.0 700 47.6 $ 14 24 it 2.0 It 2.7 it 1.5 1360 4.0 665 49.0 $ 15 24 n 2.9 II 3.5 ii 1.5 1915 3.5 994 51.9 $ 16 24 ti 2.0 II 2.6 it 1.5 1333 4.0 640 48.1 $ r 3.5 Enterotoxin recovery 2t> Lot No. Time of Dialysis Total Volume C_ DC Cat Test Doses C To xin DC 1'ox i n Enterotoxin recovery Vol. ml. Tot. Vol. Tot. ml. 2.3 609 6.0 300 49.3 % 17 24 hrs. 1.4 L. 1.8 L. 18 24 II 2.0 II 2.5 n 2.0 980 4.5 551 56.2 % 19 24 II 2.2 II 2 .70II 2.0 1100 5.0 540 49.1 % 24 24 II 6.6 II 8.64 it 2.0 3290 5.0 1730 52.5 % 25 24 II 6.0 II 7.2 1.8 3360 5.0 1440 42.9 % 26 24 II 6.6 II 8.18" 1.7 3860 4.5 1820 47.2 % 27 24 II 7.0 II 8.5 II 2.0 3500 4.5 1890 54.0 % 28 24 II 7.1 ti 8.5 ti 2.0 3550 5.5 1550 43.6 29 24 II 7.4 ti 8.8 ii 1.5 4940 4.0 2200 44.6 % 30 24 n 7.5 n 8.8 ii 2.0 3750 5.0 1760 47.0 % 31 24 it 7.6 n 9.0 II 2.0 3800 5.0 1800 47.4 % 32 24 II 7.6 II 9.1 II 2.0 3800 5.0 1800 47.4 % 33 24 II 9.8 II 9.2 II 1.5 5080 5 .0 1840 36.3 % 34 24 II 7.2 II 8.9 II 1.5 4800 5.0 1780 37.1 % 35 24 it 7J.2 II 8.9 II 1.5 4800 4.5 1980 41.3 % Note II &.) l o t s 60-64 - data recorded by Casselman (12) b) l o t s 7, 8, & 9 were dialyzed f o r 72 h r s . because of the f a i l u r e of the a i r pressure supply. % - d i a l y s i s may 27 be brought about by such undetermined f a c t o r s as the varying temperature of the water, the changing mineral constituents of the water, and the quantity of materi a l being treated at one time. In spite of these serious disadvantages, d i a l y s i s i s s t i l l considered by most workers to be - i n many cases - the least destructive and most convenient method to dispose of medium constituents, and after a l l , the average loss of s l i g h t l y more than 50 per cent of the o r i g i n a l potency i s not excessive when one considers that the t o t a l solids content has decreased some 98.34 by per cent. B. Concentration Methods Probably because of the extremely high d i l u t i o n of the toxic f a c t o r , i t has been found essential to adopt some means of concentrating the DC f r a c t i o n before i t s precipitation. a In the past, Gasselman and Wood u t i l i z e d » the Desi-vac treatment available at Connaught Medical Research Laboratories f o r the drying of f i l t r a t e s . cause of the inconvenience of packing, express But be- charges and long delays, several attempts were made to evolve a method which could be employed l o c a l l y , and which would produce r e s u l t s as good as, i f not better than, those obtained by shipping the f i l t r a t e s to Toronto. These methods included vacuum d i s t i l l a t i o n , l y o p h i l i z a t i o n , freezing, and adsorption. - 1. 28 - "Desi - vac" treatment Before the investigations of these various meth- ods of concentration are summarized, the "Desi - vac ? treat1 ment, along with t y p i c a l r e s u l t s .will he outlined. these findings, i t w i l l become apparent why concentration From other means of were attempted. The DC toxin, i n serum bottles containing 400 of f i l t r a t e , was Laboratories, the drying was shipped to the Connaught Medical Research School of Hygiene D i v i s i o n , Toronto, where done on Desi-vac equipment under the direct*, ion of Dr. A* M. F i s h e r . was ml. The dried, dialyzed type C toxin designated as "type DDC toxin". In most cases, before use, the DDC toxin was re- constituted by the addition of d i s t i l l e d water i n a quanti t y that resulted i n a f o r t y - f o l d concentration o r i g i n a l DC toxin. Invariably, a portion of the DDC ed as an insoluble f r a c t i o n ; t h i s was "RP", remain- assigned the term and the soluble f i l t r a t e , the term One of the "RS". of the c r i t e r i a for the success of the con- centration method was the reaction of the product to the acid p r e c i p i t a t i o n technique of p u r i f i c a t i o n . In general, t h i s involved the following. adjust- ed to pH 3 . 3 with 5 U HCl. The RS f r a c t i o n was The a c i d i f i e d mixture was plac- ed i n the r e f r i g e r a t o r for 3 hours, and then the f l o c c u l a r p r e c i p i t a t e (designated "p") was f i l t e r e d o f f on Whatman's 29 # 1 paper, dried at 37°C. i n a i r , or at 23°C. in*vacuum drying oven, and weighed. The f i l t r a t e (designated "FP" was adjusted to pH 7.0 with 5 N NaOH• Dilute a l k a l i (pH 8 to 9) was used to redissolve the acid p r e c i p i t a t e P, and t h i s procedure again resulted i n the formation of a soluble portion (designated "SP) and an insduble portion (designated "IP") CHART I . A summary of the various terms used i n the acid p r e c i p i t a t i o n technique of p u r i f y i n g DDC toxin. DDC toxin I reconstituted SP (solubTe) IP (insoluble) The r e s u l t s of f i v e of the major experiments i n volving Desi-vac treated DC toxin w i l l now be summarized i n various tables, i n such a way that the average decreasing weight per C.T.D. w i l l be evident, and that the average potency recoveries at the various stages can be 30 - calculated. However, i n order that the entire sequence of r e s u l t s can her stated at t h i s point - the following calculations may he made f o r C and DC toxin. C toxin: Average vol./C.T.D. 1.7 ml. average wt. /ml. .02816 gms. .% average wt. /C- T. D. « .047872 " DC toxin average v o l . /C.T.D. 4.4 ml. average wt. /ml. .00056 gms. .*. average wt. /C.T.D. =r .002464 " (The average weights per ml. are calculated from the results l i s t e d i n Table I I I , and the average volume per cat test dose - from Table IV ) Similar calculations have been made f o r the RS f r a c t i o n (Table V), a i d the SP f r a c t i o n (Table VI). Prom Table V, iiyis evident that the t o t a l s o l ids content per cat test dose of the RS f r a c t i o n has i n creased considerably; tne average weight of .002312 gms. per cat test dose i s more than twice that obtained by Casselman i n early experiments. This increase may be i n keeping with the greater destruction of potency by Desi«» vac treatment over the l a s t 2 years. The higher weight per cat test dose found i n those f i l t r a t e s ( 2 and 5) 31 TABLE V: - The r e l a t i o n o f the s o l i d content o f RS m a t e r i a l to c a t t e s t doses* wt. of RS Total * 0.5382 gms. wt ./C.T.D. CT.D.'s .000996 gms. 540 * 2.0658 «• 2300 .000902 " exp't 1)1.7605 " 660 .002670 » » 2) 3.5590 » 850 .004160 « " 3) 2.7630 '• 1400 .001975 » » 4) 1.5620 600 .002610 » » 5) 3.5920 1250 .002870 " «' " .016183 » average weight /C.T.p. = .002312 " r e s u l t s r e c o r d e d by Casselman (13) W n ich f a i l e d to respond to a c i d p r e c i p i t a t i o n , seems to substantiate this p o s s i b i l i t y . Table T J r e v e a l s , as f a r as may be by has indicated t o t a l s o l i d s content, the degree of p u r i f i c a t i o n t h a t been achieved by a c i d p r e c i p i t a t i o n . The range of 120 - 180 gamma o f t o t a l s o l i d s per c a t t e s t dose i s not too l a r g e , when one c o n s i d e r s the d i f f i c u l t i e s i n the d e t e r m i n a t i o n o f t o x i c i t y by c a t i n o c u l a t i o n s * [Further- more, the i n c r e a s e over Casselman's o r i g i n a l f i g u r e o f TABLE VI: The r e l a t i o n o f the s o l i d content of SP mate r i a l to c a t t e s t doses wt. of SP * 0 .0243 gms. * 0.1066 « wt./Q.T.D. 405 0.000060 gms. 1776 0.000060 gms. 0.0365 » 203 0.000180 «• (3) 0.0674 « 374 0.000180 « (4) 0.0375 312 0.000120 " 3070 0.000600 « .000150 " exp't(l) " Total C I . J . ' s #(2) #(5) .*, average weight /C.T.D. r e s u l t s recorded hy Casselman (13) # no p r e c i p i t a t e formed upon a c i d treatment 60 gamma, may he p a r t i a l l y e x p l a i n e d by h i s acceptance of a d i a r r h o e a l reaction as a p o s i t i v e t e s t . A l l later c a l c u l a t i o n s have been based on v o m i t i n g r e a c t i o n s only. Table VII demonstrates the removal of i m p u r i t i e s i n each of the v a r i o u s f r a c t i o n s . per The f a c t t h a t 47 cent of the potency of C t o x i n i s l o s t upon d i a l y s i s e x p l a i n s the d i f f e r e n c e between 94.49$ (100-5.51$), which i s based on s o l i d s p e r c a t t e s t dose, and 98.34$, which 33 TABLE V I I : The gradual removal of i m p u r i t i e s as r e v e a l e d by t o t a l s o l i d s content per c a t t e s t dose. fraction average wt./C.T.I). C % of s o l i d s i n C .047872 gms. 100.00 % DC .002464 " 5.51 % RS .002312 " 4.83 % SP .000150 » .31 % TABLE V I I I : The per cent o f o r i g i n a l DDC s o l i d s found i n the RP f r a c t i o n . wt. exp't of DDC % of wt. o f RP DDC (1) 2.203 gms. 0.4425 gms 20.1 % (2) 4.876 » 1.3170 « 27.0 % (3) 3.520 « 0.757 « 21.5 % (4) 1.8640 » 0.302 » 16.2 % (5) 5.3720 » 1.780 « 33.1 $ average removal -23.6 °1o i s based on the removal o f s o l i d s by d i a l y s i s , w i t h no c o n s i d e r a t i o n o f t o x i c content ( T a b l e I I I ) . The r e d u c t i o n of t o t a l s o l i d s content from 100 per cent (C t o x i n ) to 0.31 % (SP f r a c t i o n ) i n d i c a t e s a p u r i f i c a t i o n of only s l i g h t l y over 300 times. The a c t u a l removal o f i m p u r i t i e s i s much more than t h i s , but the f a c t i s masked by the r e - 34 ductionoof potency at the d i f f e r e n t stages of the process. This explains the small difference between the s o l - ids content of the DC and RS f r a c t i o n s . Because of the removal of 23.6 per cent (see Table VIII) of the s o l i d s of DDC i n the form of RP, one would expect a larger var- iation. Table VIII provides a breakdown of the f i g u r e s i n the 5 experiments which supply the information that an average of 23.6 per cent of the solids of DDC insoluble. are But the main purpose f o r t h e i r i n c l u s i o n here, i s so that the r e l a t i v e l y higher proportions of insoluble DDC i n experiments 2 and 5 can be noted. The f a c t that these 2 batches were the ones that produced no p r e c i p i t a t e indicates that the greater proportion of removed, insoluble substances may be of importance i n determining whether or not the RS f r a c t i o n w i l l react to acid treatment. Tables IX and X supply summaries of the toxin recovered i n the d i f f e r e n t f r a c t i o n s . show how those i n X were obtained. The figures i n IX The detection of only 46.7 per cent of the o r i g i n a l toxin i n RS and 3.24 per cent i n RP gives a t o t a l recovery of only 49.94 per cent of the potency i n DC. M Desi-vac w Thus, the destruction of toxin by the treatment has averaged more than 50 per cent. The fact that the average potencies of the SP (15.6 per cent) and the PP (18.4 per cent) f r a c t i o n s give a t o t a l of 34 of the o r i g i n a l 46.7 per cent of the RS 35 TABLE IX; Determination of average percentage of DC cat test doses recovered i n the various fractions. Total cat test doses i n the following: RP IP FP 203 0 300 (50) (no pp't) 0 (500) 1400 75 374 0 630 1500 600 50 312 0 120 (3200) (1250) :(CO> (no-Hp'*) 0 $.050) Totals 5700 2660 185 889 0 1050 % of DC 100 46.7 3.24 15.6 - 18.4 DC RS exp't (1) 1500 660 60 (2) 2200 (850) (3) 2700 (4) (5) *. SP The figures f o r l ost (2) and (5)nwere excluded. TABLE X: The percentage of the o r i g i n a l C toxin C.T.D.* detected i n the various f r a c t i o n s « fraction t o t a l C.T.D.'s C 12,127* DC 5,700 47 .0 RS 2,660 21.9 RP 185 1.53 SP 889 7.33 PP 1050 8.66 % of o r i g i n a l 100 This figure calculated from average recovery of 47$ toxin a f t e r d i a l y s i s . 36 f i l t r a t e , shows that the loss due to acid p r e c i p i t a t i o n i s not excessive - beingj[just s l i g h t l y over 10 per cent. Moreover, without the l i m i t a t i o n s of the cat test, i t i s quite probable that t h i s margin could be further reduced. One of the s t r i k i n g revelations of t h i s table i s the r e l a t i v e l y high potencyjof the f i l t r a t e a f t e r the acid p r e c i p i t a t e has been removed: over 18.4 per cent of the o r i g i n a l DC t i t r e i s indicated here. Because of t h i s f a c t a number of experiments to recover t h i s toxin have been attempted and t h e i r r e s u l t s w i l l be outlined l a t e r . Table X covers the same material as Table IX except that i n i t , the percent toxic content of the various fractions i s based on C material. These f i g u r e s , for the sake of c l a r i t y are repeated i n an outline chart of the process, (chart II) The recovery of 7.33 per cent of the o r i g i n a l toxin, i n a degree of purity that seems r e l a t i v e l y con/ stant, shows that t h i s means of p u r i f y i n g enterotoxin i s quite f e a s i b l e . However, the loss of over 50 per cent of the DC toxic t i t r e by Deei-vac *treatment, i s a serious v one, especially when one considers that t h i s process achieves nothing more than concentration of the f i l t r a t e . But a f a r graver f a u l t l i e s i n the f a c t thatjthe DDC toxin concentrated by t h i s means does not respond consistently to acid p r e c i p i t a t i o n * . On two occasions (experiments 2 and 5), when large batches of DDC toxin were being treated, the acid p r e c i p i t a t e d i d not form. Moreover, 37 CHART I I : The per cent of the o r i g i n a l potency of C toxin to he found i n the various f r a c t i o n s involved i n acid p r e c i p i t a t i o n . C toxin (100$) dialysis DC toxin (47$) M Desi-vac treatment DDC toxin reconstituted EP (1.53$) RS (21.9$) (pp't) (filtrate) acid ppt'n P (PPH) PP (8.66$ (filtrate redissolved Sf (7.33$) IP (0$) (filtrate) (PP't) 38 there were no variations i n procedure during these f i v e experiments that would account f o r these negative r e s u l t s . At one time i t was thought that the concentration of the toxin i n the f i l t r a t e might present a c r i t i c a l point. Weak support f o r t h i s theory may he found i n the percentages of the o r i g i n a l DC potency found i n d i f f e r e n t RS f r a c t i o n s . ( Table XI ) In experiments (2) and (5) the recovery shows a tendency to be s l i g h t l y lower than average. However, a stronger indication of t h i s possib- i l i t y was found i n the high proportion of toxin i n 3?E that would not respond to acid treatment. But, a f t e r IT f r a c t i o n s had been concentrated to 3/4, 1/2, and 1/4 of t h e i r former volume (by drying at 23'C. i n vacuo) they s t i l l f a i l e d to reveal addition precipitation. TABLE XI: The percant of the o r i g i n a l DC potency recovero ed i n i n d i v i d u a l f r a c t i o n s . Total cat test doses DC toxin Casselman(13) RS f r a c t i o n % Recovery 501 3Q1 60 exp't (1) 1500 660 44 (2) 2200 850 39 (3) 2700 1400 52 (4) 1500 600 40 (5) 3200 1250 39 39 Because of the p o s s i b i l i t y that v a r i a t i o n s i n the'besi-vac" treatment over a period of time might have changed the nature of the DDC material, and because i t was believed that the acid p r e c i p i t a t i o n phenomenon was not necessarily the demonstration of an i s o - e l e c t r i c point - small quantities of RS material were treated over a range of pH's. However, of the pH's: 1.5, 3.5, 4.5, 5.5, 7.0, 11.0, and 12.0 - only pH's: 1.5, 3.5, and revealed any t u r b i d i t y at a l l . 12.0 But the p r e c i p i t a t e s ob- tained by the pH 1.5 and 12.0 treatments revealed no t o x i c i t y (at least i t was 3e ss than 1/4 of the usual concentration) . From time to time, the extreme v a r i a t i o n i n appearance of the bottles of DDC was noted. Bottles of the same batch, which had supposedly been treated i n the same way, were returned with the DDC material i n the form of a dark brown gummy mass, voluminous f l u f f y white f l o c c u l e s , a finely-granular yellow p r e c i p i t a t e , or some intermediate form of these extremes. I n q u i r i e s as to possible variations i n Desi-vac treatment, revealed that no known changes had been i n t r o d u c e d . The data concern- ing a t y p i c a l run was as follows: "The temperature of the enterotoxin was not higher than -20 C. u n t i l 64 hours had elapsed. f At that time the temperature began to r i s e and by 70 hours had reachedk temperature of 42 C. a It was maintained at that 40 temperature f o r approximately 7 hours - that i s , u n t i l the completion of the drying. The temperature of the c i r c u l a t i n g water i n the shelves of the vacuum chamber was maintained at 12fiC. f o r the f i r s t 8-^-.hours. 10-|- hours a f t e r the commencement of the drying, the temperature of the c i r c u l a t i n g water was raised to ZO^C* and held at that temperature f o r a period of 13-^ hours;. It was then held at a temperature of 40C'c. f o r a period of approximately 24 hours, and then at 50*C» u n t i l the drying had bbeen completed. 11 (20). Nevertheless, v a s t l y d i f f e r i n g products were obtained from theisatme material, and so the problem was investigated. of weight. The f i r s t property to be studied was that The rubber caps were removed from 2 bottles containing the DDC material from 400 ml. of lot#26 DC. The dried materials were transferred immediately to beakers and weighed. The l i g h t , white, f l u f f y material weighed 2.3242 gms.; whereas, the deep cream, flaked material weighed only 0.0449 gms. Because i t was thought that t h i s marked difference might be due to the degree of dryness, the materials were placed i n the vacuum oven at 50'C. After 96 hours the reduction i n weight of the white material was 0.1455gms. material was 0.0137 gms. and that of the yellow Thus i t seemed evident that water content might be influencing the weights to a small extent, but not to a degree that would account f o r one 41 weight being 5 times that of the other. Additional exper- iments of t h i s type, involving longer drying periods, merely substantiated these e a r l i e r r e s u l t s . The next property to be investigated was of t o x i c i t y . The DDC that f r a c t i o n s were reconstituted by the addition of d i s t i l l e d water i n the usual proportions, and each material displayed a d i f f e r e n t reaction to the solvent. Most of the white material disappeared almost immediately into the water; required vigorous ution was whereas, the yellow material s t i r r i n g before evidence of any apparent. The white material was, found to contain l e s s than 1/5 dissol- i n general of the potency of the yellow; and indeed several bottles were found to contain no detectable po/ttency at a l l . Table XII summarizes the r e s u l t s of a comparison of 4 varying bottles of #26 DDC, and from t h i s i t i s apparent that the d i f f e r e n t response to a c i d treatment, the.varying pH s t a b i l i t y , and above a l l the i r r e g u l a r retention of potency, indicate that the "Desi-vac" treatment of DC toxin i s most u n r e l i a b l e . 42 TABLE XII; A comparison of four bottles of #26 DDC toxin which had undergone "Desi-vac" treatment. pH a f t e r o r i g i n a l 3 hours** C.T.D.'s C.T.D. i n RS original pH * response to pH3.5 1) 1.672gms * 8.45 PP't 6.3 80 0 2) 0.145 8.65 no pp't 6.0 80 20 3) 0.068 » 8.70 no pp't 6.0 80 20 4) 0.056 » 6.90 PP't 3.2 80 36 wt. of 400 ml. * a f t e r r e c o n s t i t i t i o n by addition of d i s t i l l e d water, following adjustment to pH 3.5. Descriptions of DDC materials: 1) white, f l u f f y , f l o c c u l e s , very voluminous. 2) l i g h t cream, flakey p r e c i p i t a t e . 3) deep cream, finely-granular powder. 4) dark brown f l a k e s , with a few almost black specks. -432. Vacuum d i s t i l l a t i o n The p o s s i b i l i t y that vacuum d i s t i l l a t i o n he u t i l i z e d was r e a l i z e d early i n t h i s i n v e s t i g a t i o n . would (18, f o r the c o n c e n t r a t i o n of e n t e r o t o x i c might filtrates Such a procedure he s i m i l a r to the i n i t i a l step used hy H e i d e l b e r g e r 19) i n the p u r i f i c a t i o n of pneumococcal polysaccharides. The experiments were performed on a v e r y small s c a l e , because i t was c o n s i d e r e d e s s e n t i a l to c o n t i n u e s h i p p i n g m a t e r i a l to Toronto f o r Desi-vac treatment (which was known to p r o v i d e f a i r l y s a t i s f a c t o r y r e s u l t s ) , and because i t was f e l t that s m a l l - s c a l e t r i a l runs would a t l e a s t i n d i c a t e whether or not the method was a p p l i c a b l e to the e n t e r o t o x i n problem. A 500-ml. C l a i s s e n f l a s k , w i t h a c a p i l l a r y p i p - e t t e f o r the c o n t r o l l e d i n f l o w of a i r , was connected through i t s condenser to a d r y i n g tube and a Hyy/ac- pump. A 500-ml. d i s t i l l a t i o n f l a s k , w i t h c o l d water f l o w i n g over i t s e n t i r e s u r f a c e , was used as the condenser, and a s m a l l , thermostatically c o n t r o l l e d water-bath maintained the d i s t i l l i n g f l u i d a t a constant temperature. was performed w i t h the eqipment The f i r s t attempted r u n assembled as above, but w i t h a water pump, i n s t e a d of the Hyvac, to reducre the a i r pressure. was However, i t was soon found t h a t the water supply n e i t h e r adequate nor constant enough to s a t i s f y require/* ments. -4-4In the f i r s t d i s t i l l a t i o n DC t o x i n was t r e a t e d u n t i l product was obtained* experiment, 400 m l . of a s l i g h t l y gummy, dark-brown 10 m l . of d i s t i l l e d water was added to t h i s , and the mixture was s t i r r e d f r e q u e n t l y , at room temperature f o r 2 hours. (RP) on The i n s o l u b l e portion was removed by f i l t r a t i o n , d r i e d a t 37*C., and weighed the f i l t e r paper; the s o l u b l e p o r t i o n to pH 3.3 w i t h IN" HCl. and and l e f t (RS) was a d j u s t e d A f l o c c u l a r p r e c i p i t a t e appeared, the a c i d i f i e d mixture was p l a c e d i n the r e f r i g e r a t o r (approximately 5°C.) f o r 3 hours. At the end of t h i s time, the p r e c i p i t a t e (P) was removed by f i l t r a t i o n , 37°C. and weighed on the f i l t e r paper. dried at The f i l t r a t e was a d j u s t e d to pH 7.0 w i t h IN NaOH, and s t o r e d i n the r e f r i g - erator. Ten m l . of pH 9.1 d i s t i l l e d water was added to p r e c i p i t a t e P on the f i l t e r paper, and passed through the f i l t e r r e p e a t e d l y f o r some 2 hours. ( L i t t l e or none of the deep brown p r e c i p i t a t e seemed to d i s s o l v e ) . After t h i s treatment only 8 m l . of f i l t r a t e were l e f t . Because one o f the c r i t i c a l comparisons o f t h i s •I method of c o n c e n t r a t i o n , as a g a i n s t . »' that of Desi-vac ment, would be the amount of m a t e r i a l treat- p r e c i p i t a t e d by a c i d treatment, the v a r i o u s p r e c i p i t a t e s were weighed, and a summary of the c a l c u l a t i o n s f o l l o w s : wt. of s o l i d s i n 400 ml.#2 DC t o x i n ( T a b l e wt. of RP s o l i d s 5)=0.0816 gms. =0.0204 * - 4-5 wt. of RS = 0.0612 gms. wt. of P s ' 0.0041 " wt. of P +• f i l t e r paper = 0.8065 « wt. of i n s o l . p + f i l t e r paper = 0.8079 » wt. of soluble P ' ' ? Possible explanations f o r t h i s phenomenon i n clude the fact that increased humidity produced a "moist" f i l t e r paper which consequently weighed more, or that the hygroscopic nature of the P f r a c t i o n d i d not permit the complete release of water molecules after the elution treatment. Calculations f o r cat doses were complicated by the fact that the amount of p which had dissolved appear- ed to be a negative value. However, f o r a s t a r t i n g point, cat doses were based on the improbable assumption that a l l the P had dissolved: wt. of s o l i d s i n 8 ml. of soluble P fraction ; 0.0041 gms. wt. of solids i n 1 ml. = 0.00052 " Wood (12) found 1 M. R. D. V 0.00006 " 1 M. R. D. should be contained i n .115 ml. However, when the approximate equivalent of t h i s quantity was inoculated into a cat, a negative reaction was obtained. La.ter inoculations of 10 and 30 times t h i s amount produced no response. P o s i t i v e cat reactions were obtained from the unprecipitated f r a c t i o n of RS, and a l though accurate calculations were impossible, i t seemed apparent that - i f the o r i g i n a l t o t a l of 133 M. R. D.'s i n 400 ml. of # 2 DC toxin - approximately 35 had been l e f t behind i n the f i l t r a t e after acid p r e c i p i t a t i o n . There- fore, i t was evident that, somewhere i n thejprocess, a considerable f r a c t i o n of thejtoxic factor had been destroyed or at least changed i n such a way that i t no longer responded to acid treatment as before. A second experiment was c a r r i e d out to determine at which stage the enterotoxin was l o s t . However, t h i s time the centrifugation, rather than the f i l t r a t i o n method of separating precipitates was used. The procedure was the same as before except that a DC toxin of s l i g h t l y higher potency was used ( M. R. D. e 2.5 ml.),and the water bath was maintained at 32*C. instead of 37°C. The centrigugation was c a r r i e d out i n a centrifuge which was cooled by a i r which had passed through a hose c o i l e d i n a solution of i c e and s a l t . By t h i s means, the temperature of the body of the centrifuge which had an o r i g i n a l reading of 16.5°C. was kept down to 21°C a f t e r one hour of operation, and 28°C. at the end of Z\ hours. In t h i s sec- ond experiment involving vacuum d i s t i l l a t i o n , the p r e c i p i t ate formed as before; i t appeared as a d i r t y yellow, f i n e l y granular p r e c i p i t a t e which r a p i d l y became f l o c c u l a r . This time the p r e c i p i t a t i n g suspension was placed i n the r e f r i g erator f o r only one hour before f u r t h e r treatment, because i t was thought that the three hour period of acid treatment might be p a r t i a l l y responsible f o r the potency l o s s . - 4-7- A summary of the r e s u l t s and calculations follows: wt. of s o l i d s i n 400 ml. of #1 DC = 0.1056 gms wt. of RP Z 0.0055 » Z 0.1001 " S 0.0066 " 0.0935 - •Ywt. of RS wt. of P wt of s o l i d s i n RS a f t e r pp't'n To p r e c i p i t a t e (P) was added 10 ml. of pH8.0 d i s t i l l e d water, and the r e s u l t i n g suspension was l e f t at room temperature f o r one hour. No attempt was made to sep- arate the insoluble f r a c t i o n by centrifugation, because i t seemed to be n e g l i g i b l e . wt. of solids i n 10 ml. of P suspension = 0.0066 gms. wt. of s o l i d s i n 1 ml. of P * 0.00066 suspension M .'• 1 M. R. D. should be contained i n 0.0909 ml. (based on Wood's c a l c u l a t i o n 1 M. R. D. Cat inoculations 0.00006 gms.) of the equivalent of t h i s amount, 5 times i t , and 10 times i t , gave negative reactions. A study was made of the RS after p r e c i p i t a t i o n : wt. of solids J i n f i l t r a t e after P removed = 0.0935 gms volume at time of test * >|5 ml. •'. wt. of solids i n 1 ml. Wood found 1 M. R. D. = 0.0187 gms .005 gms. .*. 1 M. R. D. should be contained i n When the equivalent of t h i s dose was #0.2674 mis; inoculated into a cat, a positive reaction resulted, but half of t h i s dose produced no response; that i s , the presence of at least - 4-S20 of the o r i g i n a l 100 M» R. D.'s was indicated. To the RP f r a c t i o n was added 10 ml. of d i s t i l l e d water, the suspension was well mixed, and allowed to stand at room temperature f o r 2 hours,. wt. of RP = 0.0055 gms. 1 cc. contains - 0.00055 But no cat reaction was obtained from 2.5 ml. of the r e s u l t i n g suspension; that i s , the RP f r a c t i o n contained less than 4 K. R. D.*s CHART HL: Summary of experiment 2 - the e f f e c t of vacuum d i s t i l l a t i o n on DC toxin JPC toxiQ(|.1056 gms.) (100 M. R. D.) I vacuum d i s t i l l a t i o n insoluble f r a c t i o n (RP) { .0055 gms) ("4 H. R. D.) soluble f r a c t i o n (RS) (.1001 gms.) acid pp't'n aejyt (p) .0066 gms.) ( 10 M> n» D») \ -•• filtrate (.0935) (20 M.R.D.) Thus, i n t h i s second experiment (Chart ), 20 per cent of the o r i g i n a l toxin remained unprecipitated i n the RS f r a c t i o n , no toxin was detected i n the acid p r e c i p i t ate, and approximately 80 per cent of the o r i g i n a l potency of the DC material was unaccounted f o r . 49 Because of the extreme reduction of toxin i n DC material hy vacuum d i s t i l l a t i o n , a t h i r d experiment was designed to determine whether the potency reduction i n C toxin would he as great. On two occasions, 400 ml. of C toxin and 400 ml. of DC toxin were d i s t i l l e d u n t i l they became dark gummy residues. (The achievement of an absol- utely dry product seemed impossible of the procedure; under the conditions preliminary experiments had revealed that d i s t i l l a t i o n f o r an additional 24 hours, produced no visible change i n the product.) To each of these products was added 20 ml. d i s t i l l e d water, and cat tests were performed on the resultant. These indicated that 66 2/3 per cent of the potency of the C toxin had been retained, but only 25 per cent of the DC toxin. Thus, i t was demonstrated that, under these conditions of vacuum d i s t i l l a t i o n , the impurities found i n the C toxin offerred some protection against toxin reduction. The fact that the soluble P f r a c t i o n of experiment 2 gave a p o s i t i v e Biuret, showed that the precipi t a t e was d i f f e r e n t i n i t s chemical nature as well as i n i t s i n a b i l i t y to evoke a cat reaction. Because of the aforementioned r e s u l t s , the vacuum d i s t i l l a t i o n method was put aside as being impractical under the present conditions. 50 3. L y o p h i l i z a t i o n . The l y o p h i l i z a t i o n method of concentration of toxin was investigated. The P a c i f i c F i s h e r i e s Experimental Station i n Vancouver kindly granted opportunities f o r us to use t h e i r l y o p h i l i z i n g plant; and so whenever the ap- paratus was a v a i l a b l e , material was treated i n t h i s way. F a c i l i t i e s were such that 6 l i t r e s of material could he treated at one time. This was dispensed i n 750 ml amounts into 2 l i t r e d i s t i l l a t i o n f l a s k s which possessed ground glass stopper j o i n t s f o r attachment to the drying system. The toxin was s h e l l frozen by r o t a t i o n i n a freezing mixture of brine, and then the f l a s k s were stored in the freezing room u n t i l the l y o p h i l i z a t i o n apparatus was free. Unfortunately, f a c i l i t i e s were such that only 1 f l a s k could be s h e l l frozen a t a time, and because the process required 20 minutes to 1/2 hour per f l a s k , considerable time was involved. When a l y o p h i l i z a t i o n run was to be stdrted, the f l a s k s of shell-frozen material were removed from the cold storage room, and attached with a l l possible speed to the outlets which had been previously cleaned and coated with vacuum grease. As soon as the f l a s k s were connected, the pump wasjturned on, and the pressure watched u n t i l i t was evident that there were no leaks. Because there could be no further temperature treatment of the material once i t was attached to the system, i t was most e s s e n t i a l that the reduction of pressure be very rapid; i n f a c t , i t was 51 found that i f the pressure did not approach the zero point within f i v e minutes, the material i n the f l a s k s begin to melt and bubble. would Then the process had to be stopped, and the material i n the f l a s k s refrozen. Usually when t h i s happened, one of the ground glass connections was at f a u l t , but once after lengthy investigation a IJak was found i n one of the copper pipes that connected the system to the freezing u n i t , and once, the behaviour of the pump was the source of trouble. In general, a drying period of 20 to 24 hours was found to be s u f f i c i e n t . This treatment of DC toxin usually yielded a white to cream, crusty p r e c i p i t a t e - but occasiona l l y the product would be l i g h t , and f l o c c u l a r . In general i t s reaction to resolution and acid p r e c i p i t a t i o n p a r a l l e l e d f a i r l y closely that of material treated by the method. "DeBi-vac 11 However, of 4 batches of DDCmaterial prepared by l y o p h i l i z a t i o n (see Table XIII), the average loss due to the concentration proced ure has been less than 25 per cent instead of the 50 percent reduction by "Desi-vac" treatment. Moreover, so f a r there has been no v a r i a t i o n i n the response of the r e c o n s t i t i t e d material to acid treatment - a potent p r e c i p i t a t e of yellow f l o c c u l e s has formed on a l l occasions. 52 TABLE XIII: Determination of the average percentage of Dfe cat test doses i n the various f r a c t i o n s of mate r i a l treated hy l y o p h i l i z a t i o n . Total cat test doses i n the following: DC RS RP SP I? PP 1200 900 50 250 25 200 2) 1200 1000 50 230 0 350 3) 1200 870 0 300 0 200 4) 1200 950 0 250 0 250 4800 3720 100 1030 25 1000 •t Totals % of DC 100 TABLE XIV: 77.5 2.1 . 21.5 0.5 21 The percentage of the o r i g i n a l C.T.D.*s i n C toxin detected i n the various f r a c t i o n s of l y o p h i l i z e d material. fraction t o t a l C.T.D. s 1 % of o r i g i n a l C 10, 212 100 DC 4, 800 47.4 RS -3'.: 720 36.5 RP 100 .9 SP 1, 030 10.1 IP 25 .2 PP .1, 000 9.8 53 Because i t was thought that the l y o p h i l i z a t i o n of C toxin (instead of DC) and the d i a l y s i s of the concentrated material might r e s u l t i n a lower.; potency l o s s , one hatch of f i l t r a t e was treated i n t h i s way. Six l i t r e s of C toxin, with an M.R.D. of 2.0 ml., were l y o p h i l i z e d , reconstituted by the addition of 60 ml. of d i s t i l l e d water, placed i n Visking i no c e l l u l o s e casee, and dialysed f o r 48 hours. The r e s u l t s are summarized i n TABLE XV. TABLE XV; The percentage of the o r i g i n a l C.T.D.'S i n the various f r a c t i o n s of material concentrated by l y o p h i l i z a t i o n at the C stage. fraction t o t a l C.T.D.'a % of o r i g i n a l C 3000 100 RS 1260 42 RP 200 6.7 SP 300 10.0 PP 550 18.3 Thus i t i s apparent that t h i s "short cut" i n the process does r e s u l t i n a higher retention of toxin i n the RS f r a c t i o n ; i n f a c t , there i s almost twice as much : 42 per cent instead of 21.9 per cent. cat However, the increase i n test doses does not correspond to t h i s . Nevertheless, i t i s a higher percentage of recovery (10 per cent instead of 7.33). Unfortunately though, the degree of purity of the acid p r e c i p i t a t e seems to be reduced. The calculated weight 54 of s o l i d s per cat test dose i n the SP f r a c t i o n i s 960 gamma, which i s considerably higher than the average of 150 gamma revealed by "Desi-vac" treated material. f r a c t i o n gave a p o s i t i v e Biuret. Moreover, t h i s However, i t i s quite possible that bjs attention to d e t a i l s such as d i a l y s i s time, these imp u r i t i e s could be at least p a r t i a l l y removed p r i o r to acid treatment• A study was made of the p o s s i b i l i t y that the lyop h i l i z a t i o n of u n s t e r i l i z e d C toxin might present a p r a c t i c a l means of eliminating the f i l t r a t i o n , centrifugation, and Seitzing of such large volumes of f i l t r a t e . Six l i t r e s of a 12069 alpha 3$ day culture on C medium were taken down to the F i s h e r i e s Research Station, and immediately frozen. Although the imputities seemed to o f f e r some degee of protection f o r the toxin, because over 50 per cent of the o r i g i n a l C.T.D.'s were recovered i n the Seitzed f i l t r a t e of the reconstituted material - the r e l a t i v e l y large quantity of d i s t i l l e d water that had to be added to a c h i e v e any workable solution at a l l reduced thejoriginal concentration greatly. Moreover, the re- sults after d i a l y s i s and acid treatment were much the same as above; namely, a high t o t a l solids content per cat test dose, and a p o s i t i v e Biuret reaction from the soluble part of the precipitate. Prom these r e s u l t s , i t may be seen that the lyep h i l i z a t i o n process seems to o f f e r a better means of concent r a t i o n than does the"Desi-vac" treatment. However, i t must be 55 remembered that the number of batches treated i n t h i s way has been small, and that o r i g i n a l l y the "Desi-vac" treatment also seemed to furnish r e l i a b l e r e s u l t s * 4* Comparison of Concentration Methods The r e s u l t s obtained from the various methods of concentration have been b r i e f l y compared throughout the fore- going discussions, and so f o r purposes of t h i s section - a summarizing table (XVI) of average r e s u l t s w i l l be o f f e r r e d . TABLE XVI: A comparison of the r e s u l t s obtained from 3 methods of concentrating % DC toxin recovery of toxin i n : SP f r a c t i o n SP PP wt ./C.T.D. 1.5 7.3 8.7 •wt/I50*. ? 2.0 0 0 36.5 0.9 10.1 C_ DC RS 1) 100 47 21.9 2) 100 47 3) 100 47 RP Treatment: 1) "Desi-vac" 2) Vacuum d i s t i l l a t i o n 3) L y o p h i l i z a t i o n 9.8 neg. reaction 240 * Biuret / 56 C. P r e c i p i t a t i o n .In addition to acid treatment with HCl, various prec i p i t a t i n g agents such as ethyl alcohol, acetone, methanol, and cadmium chloride were used i n an attempt*to remove the enterotoxic f r a c t i o n from RS f i l t r a t e s * A number of preliminary small-scale experiments were employed to determine the optimum conditions f o r the ethyl alcohol p r e c i p i t a t i o n of enterotoxin from RS f r a c t i o n s , and - based on these r e s u l t s - a large-scale experiment was designed to determine how the degree of purity of ethanpl precipitated material compared with that of SP. The procedure was as follows. 40 ml. of RS and 60 ml. of ethyl alcohol were cooled i n the r e f r i g e r a t o r . When both registered 5°C.» the RS material was poured slowly into the ethyl alcohol. The mixture was l e f t i n the r e f r i g e r a t o r f o r 6 hours, and then the p r e c i p i t a t e was f i l t e r e d o f f , dried, aad weighed, resuspended, and tested. The r e s u l t s are sum- marized i n Table XVII; from which i t i s evident that the ethanol-precipitated material had less than o n e - f i f t i e t h of the a c t i v i t y of the acid p r e c i p i t a t e , and that the weight of alcohol p r e c i p i t a t e per C.T.D. was more than 3 times that of jk SP per C.T.D. Thus i t i s apparent that ethanol precip- i t a t i o n either destroys enterotoxin, proportion or precipitates a greater of impurities with i t Because Sit was thought that some of the 8.66% of 57 TABLE XVII: The recovery and degree of purity of ethanol prec i p i t a t e d toxin compared to SP. RS material ethanol pp't Total weight 3.2284 gms 1.762 gms. Wt./C.T.D. 0.0025 0.0083 " 0.000150gms. 0.53$ 7 .33$ $ of o r i g i n a l toxi n recovered " 20$ SP o r i g i n a l toxin might he recovered from the PP f r a c t i o n , ethano l p r e c i p i t a t i o n s were done on a number of small quantities. Although a p r e c i p i t a t e formed each time, no potency could be detected i n i t , and a l l that the procedure accomplished was a destruction of approximately 35per cent of the PP potency. Acetone p r e c i p i t a t i o n of enterotoxin yielded s l i g h t l y better r e s u l t s than d i d ethanol. The f r a c t i o n of o r i g i n a l toxin recovered was 1.3 per cent,instead of only 0.53 per cent, and the weight per C.T.D. was 0.0052 gms. instead of 0.0083 gms. However, the procedure was s t i l l f a r i n f e r i o r to the acid p r e c i p i t a t i o n technique as regards both purity and percentage recovery of toxin. Cadmium chloride was used as a p r e c i p i t a t i n g agent upon RS material. In general, the technique of Lingood (21) was employed; the 1/3 volume of a 5$ saturated solution of cadmium chloride was used, and a pH of 6.5. A white, f l o c c - 58 u l a r , voluminous p r e c i p i t a t e was formed, and tests revealed that the active component had "been removed from the f i l t r a t e . However, residual cadmium i n washed, redissolved, and treated suspensions proved too toxic i n animal tests to permit the degree of separation of enterotoxin to he determined. Methanol p r e c i p i t a t i o n produced the best r e s u l t s of a l l the agents employed - with the exception of a c i d . A pH 4.5 buffer of, 0.5M. KH2PO4 - ISfe^HPC^ was prepared, and 10 ml. of i t , 20 ml. of methanol, and 20 ml. of RS material were c h i l l e d i n the r e f r i g e r a t o r . The methanol and buffer were then mixed, and the RS material added slowly. This mixture was then c h i l l e d at -2*0. f o r 3 hours, at the end of which time, a f i n e white p r e c i p i t a t e was evident. uged down jtjb This was c e n t r i f - i n a c h i l l e d centrifuge cup, resuspended i n c h i l l - ed pH 4.5 phosphate buffer f o r 15 minutes, and- recentrifuged. This l a t t e r step was repeated twice and then the p r e c i p i t a t e was dissolved by the addition of phosphate buffer at pH 7.4. This solution was found to contain 22 per cent of the o r i g i n a l potency of the RS material, which finding compared quite favourably with the 35 per cent demonstrated i n acid p r e c i p i t a t e . However, the C.T.D. weighed approximately 6 times the average 150-gamma-weight of SP. Because of the p a r t i a l success of t h i s experiment, t h i s p r e c i p i t a t i o n procedure was t r i e d on RS f r a c t i o n s which would not respond to acid treatment. In no case did a pred- i p i t a t e form, although a high toxin t i t r e was indicated by 59 cat tests on the f r a c t i o n . Thus i t seemed that the c o n t r o l l - ing factor of acid p r e c i p i t a t i o n also influenced of enterotoxin to methanol. the response When methanol p r e c i p i t a t i o n was attempted of the FP f r a c t i o n , a p r e c i p i t a t e formed, hut i t lacked potency. D. Adsorption It has been found that "Norit A" charcoal removes 50 per cent of the toxic potency from G toxin, 20 per cent from DC toxin, and 100 per cent from RS f r a c t i o n s . However, i n spite of many attempts, thejelution of the potent factor from the charcoal has proven unsuccessful. D i s t i l l e d water and saline at pH's of 3.5, 5.5, 7.5, 9.5, and 11.5, have proven unsuccessful at room temperature, 37'C. and 56'C. E t h y l alcohol and acetone were also i n e f f e c t i v e . cent report However, the re- (22) of similar d i f f i c u l t i e s encountered i n the elution of an a n t i b r u c e l l a factor found i n peptones from "Uori t " and the discovery that - i n t h i s case- pyridine was the successful eluting agent, sjiggest that pyridine treatment should be attempted. E. Freezing From time to time, bottles of toxic f i l t r a t e s would freeze during storage i n the r e f r i g e r a t o r , although none of the other materials around them showed any such tendency. But when one beaker containing RS material displayed a clear i c e throughout, except f o r a very small layer of bright yellowmaterial i n the bottom, the possible a p p l i c a b i l i t y of concen- 60 t r a t i o n of toxin by d i f f e r e n t i a l freezing was suggested. On three occasions, when small quantities of RS material were frozen, cat tests showed that the reduced volumes of f i l t r a t e s contained toxin i n : 20, 20, and 25 times their former concentrations. Furthermore, these concentrated RS f r a c t i o n s re- sponded well to acid treatment, and the resultant precipitates were shown to he potent. Unfortunately, the quantities invol- ved were so small that accurate calculations of weights were impossible. However, further experiments might reveal the f r - eezing method to be suitable f o r the concentration of DC mate r i a l on a large scale. IV NATURE OF TOXIN A. Potency This subject has been dealt with extensively under the various experiments concerned with the concentration of t o x i n ^ because the degree of purity - as revealed by t o t a l solids content - has been one of the p r i c i p a l c r i t e r i a f o r the success or f a i l u r e of various processes. t h i s p o i n t , i t should be s u f f i c i e n t to say that appears to be only moderately potent. Therefore, at enterotoxin Weight calculations have shown that i t has been p u r i f i e d to the point where 1 cat test dose contains 0.15 mgms. of s o l i d material. The fact that a number of p u r i f i c a t i o n experiments gave r e s u l t s that corresponded so c l o s e l y , seems to indicate that the toxin may be reaching i t s f i n a l stages of p u r i t y . The v a r i a t i o n between 60 and 180 gamma i s not large when one considers the possible 61 sources of error. The r e l a t i v e l y large amount of material, i n comparison to other toxins, that constitutes a reacting dose - may he explained by the r e l a t i v e i n s u s c e p t i b i l i t y of the cat as compared to humans. Unfoitunately, t h i s point w i l l only be s a t i s f i e d by the accurate determination of the cat to human reacting-dose-ratio. B> Non- Protein Properties A number of properties of enterotoxin have supplied evidence that the potent material i s not p r o t e i n - l i k e i n nature* The acid-precipitated material y i e l d s p o s i t i v e Mo^- l i c h and negative Biuret tests with a f a i r degree of regulari t y , and cats may be inoculated a great many times - especial- l y since the use of f i l t r a t e s prepared o^°synthetic medium before they reveal any degree of resistance to the t o x i n . Therefore, an attempt was made to demonstrate f u r ther i t s possible polysaccharide experiments. nature by means of enzyme A preliminary series of experiments using crude proteose peptone f i l t r a t e s of 12069 alpha indicated that a treatment at 37°G. f o r 4 hours worked s a t i s f a c t o r i l y for the following enzymes at their optimum pH's. pepsin 1.8 trypsin......... 8.5 caroid 7.0 takadiastase.... 7.0 However, a l t h o u g h the destruction fif alpha toxin by pepsin and t r y p s i n , but not by caroid and takadiastase, 62 TABLE XVIII? Milligrams of nitrogen i n the various f i l t r a t e s as revealed by micro-Kjeldahls* mgms ff./C.T.D. I II Average C 2.240 1.8400 2.0400 DC 0.100 0.1200 0.1100 RS 0.055 0.0620 0.0580 RP 0.032 0.0500 0.0410 SP 0.004 0.0030 0.0035 could he demonstrated - even i f exposure times were reduced to 15 minutes, negative eat tests resulted from a l l treated filtrates. Greatly d i l u t e d concentrations of enzymes and the use of DC f i l t r a t e s gave some evidence of the probable polysaccharide nature of enterotoxin, but i t was r e a l i z e d that further experiments would be valueless u n t i l p u r i f i e d '''^ enzyme preparations had been obtained. Supporting evidence f o r the non-protein nature of enterotoxin has been recently supplied by mici^Kjeldahl^ r e s u l t s which were made possible by the kind cooperation of members of the P r o v i n c i a l Department of Health in Vancouver. Laboratories The r e s u l t s of the readings on 2 d i f f e r e n t sets of f i l t r a t e s are summarized i n table XVIII. Through them are revealed the marked reduction of nitrogen content at each stage of p u r i f i c a t i o n and the f i n a l nitrogen concentration i n the SP f r a c t i o n of l e s s than 2 percent that 1 M.R.D. * 180 gamma). (based on the f a c t 63 SUMMARY 1. The constituents of a synthetic medium and the condi t i o n s required by 12069 alpha f o r good toxin product i o n have been redetermined. 2. D i a l y s i s treatment has been shown to produce a marked loss of potency, and reduction of s o l i d s i n C t o x i n . 3. Vacuum d i s t i l l a t i o n experiments indicated an almost complete loss of potency because of t h i s treatment. 4. Investigation of l y o p h i l i z a t i o n revealed that t h i s means of concentration might produce more r e l i a b l e and satisfactory r e s u l t s than "Desi-vac" treatment. 5. The freezing method of concentration was shown to be applicable to the enterotoxin problem. 6. The elution of adsorbed toxin from "Norit A" was not achieved. 7. P r e c i p i t a t i o n of enterotoxin by ethanol, methanol, acetone, and cadmium chloride was achieved, but only the methanol treatment showed any evidence that would warrant further i n v e s t i g a t i o n . 8. The soluble f r a c t i o n of acid-precipitated enterotoxin was found to have an average weight of 150 gamma per cat test dose, and a nitrogen content of l e s s than 2 per cent. 6f BIBLIOGRAPHY 1. Dack, G.M., Cary, W.E., Woolpert, 0., and Wiggers, H.» J . Frev. Med., 4, 167. 1930. 2. Pulton, P . , Br. J . Exp. Path., 24, 65, 1943. 3. Woodward, J.M., and Slane$z, L.W., J . Bact., 42, 819, 1941. 4. Kojima, T.» and Kodama, T., Kitasato Arch. Exper. Med., 16, 197, 1939. 5. Dolman, C.E., Can. J . Pub. Health, Sept 1944, 339. 6. Darrach, M«, p.2. "Studies on t h e p u r i f i c a t i o n of staph, tox." 7. Hammon, W. MeD., Amer. J . Pub. Health, 31, 1191, 1941. 8. F i l d e s , P., Richardson, G.M., Knight, B.C.J.G., and Gladstone, G.P., Br. J . Exp. Path., 17, 481, 1936. 9. Knight, B.C.J.G., Bioehem. J . , 31, 966, 1937. 10. Porter, J.R., and Pelczar, M.J., J . Bact., 41, 173, 1941. 11. Richardson, G.M., Bioehem. J . , 30, 2184, 1945. 12. Favorite, G.O., and Hammon, W.McD., J". Bact., 41, 305, 1941. 13. Casselman, W.G.B., "Studies on the preparation, p u r i f i c a t i o n , and properties of staphylococcal enterotoxin," 1947. 14. Surgalla, MLJ., and Hite, K.E., Proc. Soc. Exp. B i o l , and Med., 61, 224, 1946. 15. Surgalla, M.J., J . Inf. Dis., 81, 97, 1947. 16. Wood, J.E., "Studies on staphylococcal enterotoxin," 1947. 17. Wolf, P.A., J . Bact., 49, 465, 1945. 18. Heidelberger and Dawson, J . B i o l . Chem., 118, 61, 1937. 19. Heidelberger, J . Exp. Med, 64, 559, 1936. 20. Fisher, A.M., Personal communication to Dr. C.E. Dolman. 21. Langood, F.V., Br. J . Exp. Path., 22, 255, 1941. 22. Scherhardt, V.T., Rode, L.J., Foster, J.W., and Oglesby, G., J . Bact., 57, 1, 1949.
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